Aminoglycoside antibiotics, such as gentamicin, tobramycin, amikacin etc. are valuable antimicrobial agents. This is especially true of gentamicin which presently is widely used for treatment of infections due to gram-negative microorganisms. Great care must be taken, however, in administering aminoglycoside antibiotics since the minimum effective dosage level lies close to the threshold of toxicity. Excessive levels of aminoglycoside antibiotics, such as gentamicin in the blood of a human patient can have serious adverse consequences such as deafness and/or kidney damage. Administrations of such antibiotics at levels inadequate to destroy infectious microorganisms, can result in evolution of resistant strains of microorganism. It is therefore critical when using these antibiotics that the level of the antibiotic in the blood of a patient be carefully monitored in order to maintain an effective therapeutic dosage without exceeding the threshold of toxicity.
For assaying the blood level of gentamicin the art has developed procedures based on the fact that gentamicin inhibits the synthesis of the enzyme urease by certain bacteria. Various species of the genus Proteus, for example, are known to synthesize urease in the presence of urea. The urease enzyme attacks, in turn, the urea and destroys it by hydrolyzing it to ammonia and carbon dioxide. The presence of gentamicin in the system, however, inhibits the synthesis of urease by certain Proteus species thereby proportionately reducing the rate at which the urea is hydrolized. The concentration of gentamicin is thus inversely related to the rate of hydrolysis of urea in such a system and may be determined from measurements of the urea hydrolysis. One technique for monitoring the urea hydrolysis rate follows the reduction in the rate of increase in the pH of a culture medium due to the release of ammonia during the hydrolysis; Noone et al., Simple, Rapid Method for Assay of Aminoglycoside Antibiotics, Lancet July 3, 1971, pages 16-19. See also ibid. Dec. 2, 1972, pages 1194-1195; ibid. Jan. 6, 1973, pages 49-50, and ibid. Feb. 10, 1973, pages 315-317. It is also known to follow hydrolysis of urea by the enzyme urease by using test media containing urea labeled with radioactive carbon 14 and measuring the evolution of radioactive carbon .sup.14 CO.sub.2. McDonald et al., Urease: A Sensitive and Specific Radiometric Assay, Enzymologia, Vol. 42, pages 1-9; DeBersaques, A Micromethod for Urease, Liquid Scintillation Counting, Vol. 3, M. A. Crook, ed., Heyden, pages 303-306. A particularly advantageous approach for the assay of gentamacin in blood plasma is detailed in copending U.S. patent application Ser. No. 452,264 now U.S. Pat. No. 3,948,729, owned by the common assignee and incorporated herein by reference. These previously reported bioassays of gentamicin concentration have utilized strains of the species Proteus mirabilis.
Microbiological assays of aminoglycoside antibiotic levels are subject to the difficulty that aminoglycoside antibiotics like gentamicin are most commonly administered in conjunction with one or more additional antibiotics. Since the mircroorganisms conventionally utilized for such assays can be, and often are, sensitive to the additional antibiotics present in the patient's blood, the results of the assay may be distorted due to the fact that the additional antibiotic may kill the microorganism; thereby decreasing the synthesis of the enzyme urease and consequently reducing the hydrolysis of ammonia so that the assay erroneously indicates a higher than actual level of aminoglycoside antibiotic in the patient's blood. Representative antibiotics commonly administered in conjunction with aminoglycoside antibiotics such as gentamicin include chloramphenicol, ampicillin, tetracycline, carbenicillin, furadantin, sulfisoxazole, cephalothin and clindamycin. While deactivation of the foregoing antibiotics in order to prevent their interfering with the aminoglycoside antibiotic assay is theoretically possible, it is ordinarily difficult, especially since the testing laboratory may not have been told of the complete antibiotic regimen used on the patient. Accordingly, it is important to have an assay for determining the level of an aminoglycoside antibiotic, such as gentamicin, tobramycin, amikacin, etc., in blood serum or plasma which is not susceptible to interference from other antibiotics commonly administered in conjunction with the aminoglycoside antibiotics.